7. Project Summary/Abstract. We propose 3 specific aims to elucidate mechanisms regulating IgH class switch recombination (CSR) and Ig variable region exon somatic hypermutation (SHM). The major premise guiding Aims 1 and 2 is that chromatin loop extrusion plays a key role in promoting and regulating CSR and SHM. The major premise guiding Aim 3 is that intestinal Peyer's Patch (PP) GC responses involve expansion of rare BCR clonotypes that acquire high level intrinsic SHMs. These premises are supported by a wealth of strong published and preliminary data derived in large part from powerful new technologies that we developed during the current funding period. These assays, which will be used for ongoing studies, include a high throughput CSR assay (CSR-HTGTS) with sensitivity and resolution far beyond that of prior assays, an exceptionally high-resolution chromatin- interaction assay (3C-HTGTS), a rapid ES cell-based V(D)J knock-in passenger allele system to test impacts of sequences and cis-acting regulatory elements on SHM in germinal center (GC) B cells, and a repertoire/SHM sequencing method to study SHM features across complete GC IgH and IgL repertoires. Our first aim will test the hypothesis that cohesin-mediated loop extrusion generates large IgH loops anchored downstream by the 3'IgH regulatory region (3'IgHRR) and upstream by donor S within which cytokine/activator-induced I region promoter transcription through S regions generates impediments to ongoing extrusions resulting in directional synapsis of S and acceptor S regions for orientation-specific CSR. Mechanisms downstream of synapsis that contribute to orientation-specificity of CSR will be elucidated by testing ability of designer nuclease-targeted S region DSBs, divergent S region sequences, and inverted S region transcription units to mediate this activity. Aim 2 proposes to test, for both IgH and IgL genes, the hypothesis that chromatin loop extrusion juxtaposes downstream enhancers with V(D)J exons to make them a privileged SHM location. A key experimental approach will be to use our passenger system to assay germinal center GC B cells with matched IgH or IgL productive and passenger alleles, derived from physiological V(D)J rearrangements, for effects of test mutation(s) on the passenger allele for effects on SHM and loop extrusion. Aim 3 will test the physiological impact of recurrent antibody clonotypes expression in PP GC B cells that we discovered through endogenous repertoire/SHM sequencing studies that also revealed some recurrent clonotypes to be microbiome-dependent. To elucidate physiological implications, we will further test the hypothesis that these recurrent antibodies may be involved in an immune response against gut microbiota or other gut antigens by characterizing their antigen recognition properties and by their ectopic or ablated expression in mouse models. The proposed work should advance our understanding of key antibody maturation mechanisms and may also lead to new insights into potential roles of PP GC-derived antibodies with respect to involvement in normal physiology or disease.